1. Field of the Invention
This invention relates to monoclonal antibodies specific to the antigenic determinants of carcinoembryonic antigen (hereinafter referred to as CEA), a process for producing such antibodies and the use of such antibodies.
2. Prior Art
CEA is a well known cancer-specific fetal antigen which is a kind of glycoprotein having a molecular weight of about 200,000.+-.80,000 and a ratio of sugar to protein of about 1:1. The presence of CEA as a cancer-specific antigen in adenocarcinoma of human digestive tract was previously reported by Gold and Freedman [J. Exp. Med., 121, 439 (1965) and ibid., 122, 467 (1965)]. Although the significance of CEA for clinical uses such as, for example, a marker for detecting and treating human cancer by radioimmunoassay of CEA in blood as well as for various fundamental medical studies is widely recognised in the art, there are certain CEA-related normal antigens which make the cancer-specificity of CEA unclear because of their cross-reactivities with CEA. Examples of such CEA-related antigens include non-specific cross-reacting antigen (hereinafter referred to as NCA) which is a kind of glycoprotein having a molecular weight of about 80,000.+-.30,000 and a sugar content of about 1:1 and which is found, for example, in human lung and spleen [Proc. Natl. Acad. Sci. USA., 69, 2492 (1972 )] and normal faecal antigen (hereinafter referred to as NFA) which may further be classified into NFA-1, NFA-2 and normal faecal cross-reacting antigen (hereinafter referred to as f-NCA) as follows:
NFA-2 is a kind of glycoprotein having a molecular weight of about 200,000.+-.50,000 and a ratio of sugar to protein of about 1:1. Its antigenic and physicochemical characteristics are very similar to those of CEA.
Both NFA-1 and NFCA appear to be decomposition products of NFA-2. NFA-1 is a low molecular weight antigen having a molecular weight of about 20,000 to 30,000 and a sugar content of about 13%, and NFCA is a kind of glycoprotein having a molecular weight of about 80,000.+-.30,000.
In addition there is an antigen designated as faecal non-specific cross-reacting antigen (hereinafter referred to as f-NCA) in normal human faeces, of which the antigenic properties are substantially the same as those of the above-mentioned NCA. f-NCA is cross-reactive with CEA, NFCA and NFA-2 [Cancer Res., 41, 713-720 (1981)]. Thus, there are 4 types of CEA-related antigens of normal human faeces origin, which are of interest in this regard.
In order to use CEA as a cancer marker with better results, it is necessary to distinguish CEA clearly from such CEA-related antigens. For this purpose, it has hitherto been desired to provide an anti-CEA antibody having a definite specificity to the antigenic determinants of CEA. However, various polyclonal anti-CEA antibodies of the known types have commonly the disadvantage of unclear reaction specificity because such polyclonal anti-CEA antibodies comprise mixtures of various different antibodies which may react with almost all of the numerous antigenic determinants on the CEA molecule. For this purpose of overcoming such a difficulty, various attempts have hitherto been made to provide monoclonal anti-CEA antibodies because it has been believed in the art, for example, as follows:
(1) Monoclonal anti-CEA antibody may be specific to a sole antigenic determinant when prepared in conventional manner by fusing the cells and thus may have an uniform reactivity with antigen.
(2) It may be possible to produce a large amount of antibody having the desired uniformity by propagating such monoclones; and
(3) It may be possible to provide many kinds of monoclones exhibiting a wide variety of specificity as in the case of polyclonal antibodies.
Thus, the production of monoclonal anti-CEA antibodies is disclosed, for example, by Accolla, R. S. et al, Proc. Natl. Acad. Sci. USA., 77, 563 (1980); Mitchell, K. F., Cancer Immunol. Immunother., 10, 1 (1980); Rogers, G. T. et al, Br. J. Cancer, 43, 1 (1981); Kupchik, H. Z. et al, Cancer Res., 41, 3306 (1981) etc. However, it has not yet been clarified which known monoclonal anti-CEA antibodies are specific to which antigenic determinants of the CEA molecule which has a very complicated antigenic structure.
Namely these reports disclosed in summary the following findings as to the reaction specificities of the monoclonal anti-CEA antibodies which they obtained:
(1) Accolla et al reported that the antibodies obtained from two hybridoma clones reacted very weakly with NGP (which is believed to be equivalent to NCA as hereinbefore described) and strongly with CEA and that the reactions of these two antibodies with CEA were not competitively inhibited in relation to each other indicated that they react with different antigenic determinants on the CEA molecule, respectively.
(2) Mitchell reported that a monoclonal anti-CEA antibody which he found reacted with CEA but not with NCA and that its reaction with CEA was not inhibited by a polyclonal goat anti-CEA antibody.
(3) Rogers et al merely reported that a monoclonal anti-CEA antibody reacted weakly with a CEA extracted from tumours but strongly reacted with another CEA extracted from patients' sera.
(4) Kupchik et al analysed the reaction specificty of only one preparation of monoclonal anti-CEA antibody which was produced by one clone out of 9 cloned hybridomas in comparison with that of a polyclonal goat anti-CEA antibody and reported that such monoclonal anti-CEA antibody reacted with a variant subpopulation of CEA molecules which were reactive with the polyclonal anti-CEA antibody.
No further clarification of the reaction specificities of these known monoclonal anti-CEA antibodies has been reported.
Meanwhile, we have found the presence of certain CEA-related antigens in normal adult faeces viz. NFA-1, NFA-2 and NFCA as hereinbefore described and have succeeded in isolating them [Japanese Patent Application as laid open to public inspection as Kokai Koho 46819/81; Cancer Res., 41, 713-720 (1981); and Molecular Immunology, Vol. 19, No. 3, pp. 399-406 (1982)] and moreover, as a result of investigating the antigenic structure of the CEA molecule using such CEA-related normal antigens, we have been able to classify many antigenic determinants on the CEA molecule, for example, into the following 5 types:
(1) Individual specific antigenic determinant:
Specifically present in a given CEA sample used as the immunising antigen and not present in any other human CEA sample.
(2) CEA-specific antigenic determinant:*
Commonly present in various human CEA samples and not in CEA-related normal antigens such as NFA and NCA. This determinant has the highest cancerspecificity.
(3) NFA-1 common antigenic determinant:*
Commonly present in CEA, NFA-2 and NFA-1. One of the main determinants on the CEA molecule.
(4) NFCA common antigenic determinant:*
Commonly present in CEA, NFA-2, and NFCA. One of the main determinants on the CEA molecule.
(5) NCA common antigenic determinant:*
Commonly present in CEA, NFA-2, NFCA and f-NCA. One of the widely distributed determinants of CEA and CEA-related antigens. FNT [* (2) to (5) c.f. Cancer Res.,41, 713-720 (1981)].